Plasminogen-like growth factor (PLGF) was first identified as a heparin-binding molecule secreted by a human fibroblast cell line, M426, and is mitogenic towards several epithelial cell types. Purified epithelial growth factor consists of three molecular wild type species of 90, 60, and 30 kilodaltons (kDa). Neutralizing antisera was raised that could effectively block the mitogenic action of this growth factor. We have obtained protein sequencing data and found a high degree of similarity to a recently cloned hepatocyte growth factor (HGF). cDNA clones were isolated from an M426 library and the DNA sequence obtained was essentially identical to HGF. Furthermore, transient expression of the PLGF coding region in COS-1 cells had led to the production of mitogenically active growth factor that could also be immunoprecipitated by our neutralizing antibodies. Our cDNA clones have identified an open reading frame of 2184 base pairs which may encode a protein of 728 amino acids. Computer search through a protein data bank revealed interesting similarity to a spectrum of serine proteases among those tissue plasminogen activators. Based on this homology, we were able to delineate PLGF into a precursor monomeric 90-kDa species which upon cleavage may generate a 60-kDa N-terminal alpha subunit and a 30-kDa C-terminal beta subunit with both forms possibly linked by disulphide bridge(s). Northern analysis of RNA from various cell lines has identified multiple forms of transcripts which may encode part of the mature PLGF and is the focus for future studies.